Step I: White Paper Application

Application Guidelines 1. The application should be submitted electronically per requirements via the web site of any of the NIAID Genome Sequencing Centers for Infectious Diseases. Include all attachments, if any, to the application. 2. There are no submission deadlines; white papers can be submitted at anytime. 3. GSC personnel at any of the three Centers can assist / guide you in preparing the white paper. 4. Investigators can expect to receive a response within 4-6 weeks after submission. 5. Upon approval of the white paper, the NIAID Project Officer will assign the project to a NIAID GSC to develop a management plan in conjunction with the participating scientists.

White Paper Application

Project Title: Sequence Analysis of the Genus Acinetobacter Authors: Primary Investigator Contact: Name Patrice COURVALIN Position Professor, Head of the "Unit des Agents Antibactriens" Institution Institut Pasteur Address 25, rue du Docteur Roux State Paris, France ZIP Code 75015 Telephone 33 1 45 68 83 20 Fax 33 1 45 68 83 19 E-Mail Patrice.courvalin@pasteur.fr 1. Executive Summary (Please limit to 500 words.) Provide an executive summary of the proposal. The genus Acinetobacter is a taxonomically highly heterogeneous group of biochemically versatile organisms which occupy different natural ecosystems and play an increasing causative role in opportunistic human infections. Currently, the genus consists of 27 validly named species, seven provisionally termed genomic species and a high number of strains of an as-yet unknown taxonomic status. The taxonomy of the genus still suffers from many drawbacks such as the confusing nomenclature of provisional taxa, unclear taxonomic status of same (tentative) species, a high number of as-yet unclassified strains and the lack of reliable practical identification methods. The most medically important species of the genus is Acinetobacter baumannii, a worrisome nosocomial pathogen responsible for infections such as pneumonia, infections of the urinary tract, bloodstream, and skin and soft tissue. A. baumannii constitutes a major public health problem owing to its propensity to develop resistance to numerous antimicrobial agents and to spread among patients in hospitals. Extensively drug-resistant or even pandrug-resistant strains are emerging in clinical settings worldwide and mostly belong to a few successful epidemic global lineages. The aim of the proposal is to obtain full genome sequences of 114 strains covering the breadth of the known diversity of the genus Acinetobacter. These will include 22 strains of A. baumannii, 59 strains representing all other validly named Acinetobacter species and 33 strains representing tentative novel species (one to three per species). Sequence data will
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be used to: 1) improve the taxonomy of Acinetobacter including the description of novel species 2) clarify the phylogenetic structure of the genus 3) generate accurate tools for identification of Acinetobacter at the species level 4) characterize the accessory genome of A. baumannii and other species with an emphasis on mobile genetic elements such as plasmids, insertion sequences, integrative conjugative elements and genomic islands 5) define the resistome (i.e., genes and genetic elements associated with antimicrobial resistance) of A. baumannii by comparing the genomes of multidrug-resistant and wildtype susceptible strains 6) determine if Acinetobacter species or as-yet unclassified strains other than A. baumannii could represent a potential reservoir of resistance genes. Of particular interest is the question: has A. baumannii acquired the efflux systems implicated in multiple drug resistance from other Acinetobacter species? 7) complete the knowledge on the characterization of the n-alkane metabolizing enzymes in various species of Acinetobacter 8) obtain information on the presence and precise structure of resistance genomic islands and on the distribution of putative virulence genes in various species.

2. Justification Provide a succinct justification for the sequencing or genotyping study by describing the significance of the problem and providing other relevant background information. This section is a key evaluation criterion. 1. State the relevance to infectious disease for the organism(s) to be studied; for example the public health significance, model system etc. Public health importance. A. baumannii is a nosocomial pathogen involved in severe infections including ventilator-associated pneumonia, skin and soft-tissue infections, wound infections, meningitis, urinary tract infections, and bloodstream infections. A. baumannii is a major human nosocomial pathogen mainly due to the fact that (i) it is highly prevalent in many hospitals and (ii) it is able to develop resistance to multiple antibiotic classes including broad spectrum -lactams, third generation cephalosporins (1), carbapenems (2), aminoglycosides (3, 4, 5, 6), fluoroquinolones, tigecycline, and also colistine (7), currently the most effective agent to combat carbapenem-resistant A. baumannii strains. These multiresistant strains are of great concern, in particular in
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intensive care units, where they are responsible for outbreaks difficult to control and are associated with a high morbidity and a prolonged length of hospital stay. A. baumannii can persist since it is able to adhere to biological and abiotic surfaces and to form biofilms. The emergence and global dissemination of A. baumannii indicate the successful adaptation of this pathogen to the 21st century hospital environment (8). Apart from A. baumannii, other species of the genus have been recognized as clinically relevant agents involved mostly in nosocomial infections. These, in particular, include the newly described species Acinetobacter pittii and Acinetobacter nosocomialis (9), which are related to and have similar behavior in hospitals as A. baumannii, followed by A. lwoffii (that is, after A. baumannii, the second most frequent species isolated from humans although it has lower pathogenic potential compared to the above three species) (10), A. ursingii (responsible for a high proportion of Acinetobacter bloodstream infections) (11) and A. junii (shown to be responsible for nosocomial outbreaks) (12). Some other Acinetobacter species (A. calcoaceticus, A. haemolyticus, A. schindleri, A. parvus, A. radioresistens, A. beijerinckii, A. gyllenbergii, A. bereziniae, A. guillouiae, A. soli, genomic species 9, 13-17BJ, 15TU) as well as yet not classified strains have been occasionally reported as the causative agents of human infections (13, 14, 15). The real prevalence in clinical specimens as well as the pathogenic potential of different Acinetobacter spp. is largely unknown mainly owing to the lack of reliable and convenient identification methods applicable in routine laboratories and to the limited knowledge on the properties responsible for the pathogenicity and epidemic behavior of these organisms. 2. Are there genome data for organisms in the same phylum / class / family / genus? What is the status of other sequencing / genotyping projects on the same organism including current and past projects of the NIAID GSC? Provide information on other characteristics (genome size, GC content, repetitive DNA, pre-existing arrays etc.) relevant to the proposed study. Have analyses been performed on the raw data already generated/published? If additional strains are proposed for a species, please provide a justification for additional strains? The complete genomes of 6 A. baumannii and 2 Acinetobacter sp. have been sequenced: A. baumannii AYE, Accession number NC_010410 A. baumannii SDF, Accession number NC_010400 A. baumannii AB307-0294, Accession number NC_011595 A. baumannii AB0057, Accession number NC_011586 A. baumannii ACICU, Accession number NC_010611 A. baumannii ATCC17978, Accession number NC_009085 Acinetobacter sp. ADP1, Accession number NC_005966 Acinetobacter sp. DR1, Accession number NC_014259 In addition, 27 non-annotated genomes of A. baumannii, 3 of A. radioresistens, 3 of A.
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lwoffii, 3 of A. pittii, 3 of A. calcoaceticus, 2 of A. nosocomialis, 1 of A. junii, 1 of A. johnsonii, 1 of A. haemolyticus, 1 of A. bereziniae, 1 of A. parvus, 1 of A. ursingii, and 3 of Acinetobacter sp. are also available. Investigated Acinetobacter genomes range from 3.1 to 4.1 megabases in size. 3. If analyses have been conducted, briefly describe utility of the new sequencing or genotyping information with an explanation of how the proposed study to generate additional data will advance diagnostics, therapeutics, epidemiology, vaccines, or basic knowledge such as species diversity, evolution, virulence, etc. of the proposed organism to be studied. The genome data available indicate a great diversity in A. baumannii due to acquisition of virulence and resistance genes, in particular located in resistance islands of various sizes (16, 17). Therefore, sequencing of a larger number of members of the genus will provide additional information in the classification, evolution and physiology of Acinetobacter as well as in the mechanisms for acquisition of genes as detailed below. Due to difficulty in identifying Acinetobacter at the species level, knowledge on the biology, pathogenicity, or ecology of Acinetobacter remains largely unknown. The sources and modes of spread of these organisms as well as the source and the mode of acquisition of resistance genes are only partially known. A. calcoaceticus, A. baumannii, A. pittii, and A. nosocomialis are grouped within the so called A. calcoaceticus-A. baumannii (ACB) complex which represents a genomically and phenotypically distinct clade within the genus (9). Of these species, A. baumannii is primarily associated with human diseases but new studies indicate that also A. pittii and A. nosocomialis may be relatively prevalent in hospitalized patients (18). Furthermore, additional strains of the ACB complex isolated from humans have been reported (19), which do not belong to any of the four species and may thus either represent new species or result from recombination events between the genomes of known species. Species of the ACB complex seem to notably differ from each other in their biological, physiological, and pathological characteristics but also in their antimicrobial susceptibility and mechanisms of resistance to antibiotics. Thus, their identification at the molecular level is crucial. There are only a few data on the mobile genetic elements present in Acinetobacter. Although characterization and typing of resistance plasmids have been performed in A. baumannii (20), sequencing and analysis of plasmids in other species of Acinetobacter could reveal putative transfer of these elements to A. baumannii and other clinically relevant species. Finally, diversity seems to exist within A. baumannii since analysis of the available data indicates that various types of resistance islands are present in this species suggesting that the genome of A. baumannii has evolved, at least in part, through acquisition and loss of chromosomally-encoded resistance genes (21).
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Thus, study of the full genomes of various Acinetobacter species will provide important information concerning (i) improving the taxonomy and knowledge on phylogenetic structure of genus Acinetobacter, (ii) identification of the strains at the species level, (iii) presence of resistance genes in the non-A. baumannii spp. that could be acquired by A. baumannii or vice-versa, (iv) the nature of mobile genetic elements (such as plasmids, transposons, genomic islands) potentially responsible for the circulation of these genes and their spread among members of the Acinetobacter genus, and (v) characterization of putative new resistance islands and genes. Additionally, results from genome sequencing of A. baumannii strains originating from various parts of the world may provide an insight into the diversity of the population structure of this species. Several hydrocarbon-degrading bacteria have been used as model organisms to elucidate the biochemistry and the genetic basis of hydrocarbon-degrading pathways (22). However, there are only a few data on the regulation of genes encoding alkane utilization. It has been demonstrated that Acinetobacter sp. strains ADP1 and M-1 as well as Acinetobacter venetianus VE-C3 are able to degrade this molecule (2327). The organization of the genes implicated in alkane degradation is different in VE-C3 and ADP1/M-1. Analysis of the genome of various species of Acinetobacter will allow us to genetically characterize the nalkane-metabolizing enzymes in other species of Acinetobacter and to better understand the metabolism of very long chain n-alkanes in microorganisms and its regulation.

3. Rationale for Strain Selection 4. Provide the rationale behind the selection of strains and the number of strains proposed in the study. The focus of the program is on potential agents of bioterrorism or organisms responsible for emerging or re-emerging infectious diseases. Non-select agents or non-pathogenic organisms will be considered when they can provide insight into these scientific areas. Over the past decade, A. baumannii has emerged as a major public health problem, essentially because of its propensity to develop resistance to numerous drugs. In addition to intrinsic resistance mainly due to low permeability of the outer membrane, basal level expression of certain efflux pumps and chromosomally encoded cephalosporinases, A. baumannii is able to easily acquire genetic elements such as plasmids, transposons, and integrons. Genomic information available to date indicates both conservation and acquisition of elements, in particular resistance islands. Sequencing of the genome of other species will give information on the putative transfer of elements between species of
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Acinetobacter and of the extent of horizontal transfer of genetic information with other genera. Furthermore, the availability of genomic information on the diversity of the genus will be critical to definitely assign every reference strain to a given species. To these ends, we propose to sequence the genomes of 114 strains selected carefully to cover the breadth of the known taxonomic diversity of the genus Acinetobacter. These include 22 strains of A. baumannii, 59 strains representing all other validly named Acinetobacter species (www.szu.cz/anemec/Classification.pdf) and 33 strains that are representative of 16 unnamed genomic species or tentative novel species. The validly named species are represented by the respective type strains while each of the unnamed genomic species or tentative novel species is represented by a reference strain that was selected based on polyphasic analysis of multiple strains belonging to the given taxon. Only the type strain for species without known clinical relevance is included. The 21 additional strains of A. baumannii represent different genotypes of this most important species as indicated by multilocus sequence analysis and other typing methods (28). In the case of clinically relevant species other than A. baumannii, one or two additional strains have been included, if available, to approximate intraspecies variations. This is important for the development of sequence based identification methods and for defining the core/ accessory genome of different species. The additional strains were selected to differ from the corresponding type strains in genotypic/phenotypic properties and in origin (e.g. clinical versus environmental) as much as possible. Publicly available genome sequences of such a strain collection will provide researchers with a unique and comprehensive reference dataset than can be used in future studies focused on the taxonomy, population genetics, epidemiology, antimicrobial resistance, pathogenicity or ecology of the genus Acinetobacter. For some of these species, identification by a phenotypic approach is very difficult or not possible. A. bereziniae, A. guillouiae, A. ursingii, and A. schindleri have been isolated from human clinical isolates (11, 29). A. guillouiae strains have also been isolated from different environmental sources (29). Bacteremia due to A. ursingii have been described (30). Thus, these species could represent nonnegligible opportunistic pathogens and precise assessment of the genus Acinetobacter at the species level is necessary to determine the source(s) and the mode(s) of transmission of these strains in order to prevent nosocomial infections and to evaluate the clinical significance of each isolate.

4a. Approach to Data Production: Data Generation 5. State the data and resources planned to be generated. (e.g draft genome sequences, finished sequence data, SNPs, DNA/protein arrays generation, clone generation etc.) We will generate draft de novo genome sequences for all proposed strains using the Illumina HiSeq platform. Many of these genetic elements contain multiple repetitive regions, such as transposable elements, making assembly and localization of these genes difficult. To overcome this problem, we will use large jumping library insert sizes (~3-7 kb), which will enable us to scaffold over the repeated elements.

4b. Approach to Data Production: Data Analysis 6. Briefly describe the analysis (value-add) envisioned to be performed subsequently by the community and the potential to develop hypotheses driven proposals given the datasets and resources produced by this work. Additional genomic sequencing information will provide an important resource to further ongoing investigations currently limited by available sequences. Analysis of the genome of A. baumannii of distant sources will bring information on putative geographic specificities.

5. Community Support and Collaborator Roles: 7. Provide evidence of the relevant scientific communitys size and depth of interest in the proposed sequencing or genotyping data for this organism or group of organisms. Please provide specific examples. Depth of interest of scientific community. Currently, there is an increase of reports on
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genome sequencing of A. baumannii (at least four articles in 2011) responsible for epidemics of nosocomial infections suggesting an important interest in this genus. Size of scientific community. More than 450 articles have been published from January 2011 on Acinetobacter (medical impact, diagnostics, genome sequencing, antimicrobial resistance). The International Symposium on the Biology of Acinetobacter takes place every four years (the last was held in Roma in September 2010). The aim of these meetings is to discuss all aspects of the genus Acinetobacter. Readiness of scientific community to use data. It has been recently demonstrated in other bacterial genera that whole genome sequencing (i) provides insight into the nature of the genetic changes between isolates, (ii) determines the relative importance of SNPs and mobile genetic elements on the evolution of bacteria, and (iii) is an important tool to elucidate the evolutionary processes responsible for the rapid development of antibiotic resistance. We expect that the proposed project will allow a better understanding of the development of antibiotic resistance in this pathogen. 8. List all project collaborators and their roles in the project Alexandr Nemec (National Institute of Public Health, Prague, Czech Republic): selection and provision of strains, taxonomic analysis. Dominique Clermont (Collection de lInstitut Pasteur): selection and provision of strains, taxonomic analysis. Chantal Bizet (Collection de lInstitut Pasteur): selection and provision of strains, taxonomic analysis. Patrice Courvalin (Unit des Agents Antibactriens, Institut Pasteur): selection of strains, analysis of resistance genes and of their genetic basis. Thierry Lambert (Unit des Agents Antibactriens, Institut Pasteur): selection and provision of strains, analysis of pathogenicity and metabolism genes. Bruno Prichon (Unit des Agents Antibactriens, Institut Pasteur): selection of strains, analysis of resistance genes and of their genetic basis. Management of the sequencing project. The sequence project will be managed by Michael Feldgarden and Cheryl Murphy of the Broad Institute. Advisory Committee: Sylvain Brisse Institut Pasteur, Plate-forme de Gnotypage des Pathognes et sant
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publique, Paris, France Kevin Towner - Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom Ulrike Gerischer Institut of Microbiology and Biotechnology, University of Ulm, Germany 9. List availability of other funding sources for the project. None currently available

6. Availability & Information of Strains: 10. Indicate availability of relevant laboratory strains and clinical isolates. Are the strains/isolates of interest retrospectively collected, prepared and ready to ship? Note: If samples are prospectively prepared the GSC can provide protocols and recommendation based on the Centers past experiences. The samples must however meet minimum quality standards as established by the Center for the optimal technology platform (sequencing/ genotyping) to be used in the study. The strains have been retrospectively collected from the National Institute of Public Health (Prague, Czech Republic) and from the "Collection de l'Institut Pasteur". All the strains used in the project will be available in the "Collection de l'Institut Pasteur". Genomic DNA will be prepared according to GSC recommendations for preparation. 11. Attach relevant information, if available in an excel spreadsheet for multiple samples: e.g
Name Identifier Material type (DNA/RNA/Strain) Genus Species Specimen / Strain Isolation source Isolated from Select agent status International permit requirement BEIR/ATCC repository accession number Other public repository location Other public repository identifier Sample providers name Sample providers contact

12. What supporting metadata and clinical data have been collected or are planned on being collected that could be made available for community use? See Tables 1 and 2. 7. Compliance Requirements:
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7a. Review NIAIDs Reagent, Data & Software Release Policy: NIAID supports rapid data and reagent release to the scientific community for all sequencing and genotyping projects funded by NIAID GSC. It is expected that projects will adhere to the data and reagent release policy described in the following web sites. http://www3.niaid.nih.gov/LabsAndResources/resources/mscs/data.htm http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-013.html Once a white paper project is approved, NIAID GSC will develop with the collaborators a detailed data and reagent release plan to be reviewed and approved by NIAID. Accept Decline

7b. Public Access to Reagents, Data, Software and Other Materials: 13. State plans for deposit of starting materials as well as resulting reagents, resources, and datasets in NIAID approved repositories. Sequencing projects will not begin until the strain is deposited into NIAID funded BEI repository (http://www.beiresources.org/). This includes web based forms are completed by the collaborator and received by the NIAID BEI (http://www.beiresources.org/). All the strains used in the project will be available in the "Collection de l'Institut Pasteur". All sequences and read files generated under this proposal will be submitted to the Short Read Archive at NCBI/NLM/NIH on a weekly basis. These data will also include information on templates, vectors, and quality values for each sequence. Genome assemblies will be made available via GenBank and the Broad web site. Assembled contigs and scaffolds will be deposited in the Whole Genome Shotgun (WGS) section of GenBank, http://www.ncbi.nlm.nih.gov/Genbank/wgs.html, within 45 calendar days of completing assemblies. If it is determined that the final assembly can be significantly improved, an updated record will be deposited in the appropriate part of GenBank when complete. Annotation data will be made available via GenBank and the Broad web site after consistency checks and quality control have been completed by the GSCID and collaborators. Assuming no significant errors are detected during the validation process, annotation data will be released within 45 calendar days of being generated.

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7c. Research Compliance Requirements Upon project approval, NIAID review of relevant IRB/IACUC documentation is required prior to commencement of work. Please contact the GSC Principal Investigator(s) to ensure necessary documentation are filed for / made available for timely start of the project.

Investigator Signature:

Investigator Name: Patrice Courvalin

Date

References 1. Heritier, C., L. Poirel, P. Nordmann. 2006. Cephalosporinase over-expression resulting from insertion of ISAba1 in Acinetobacter baumannii. Clin. Microbiol. Infect. 12: 123-130 2. Mussi M.A., A.S. Limansky, A.M. Viale. 2005. Acquisition of resistance to carbapenems in multidrug-resistant clinical strains of Acinetobacter baumannii: Natural insertional inactivation of a gene encoding a member of a novel family of beta-barrel outer membrane proteins. Antimicrob. Agents. Chemother. 49:1432-1440. 3. Joly-Guillou, M.L., D. Decr, J.L. Herrman, E. Bourdelier, E. Bergogne-berezin. 1995. Bactericidal in vitro activity of beta-lactams and beta-lactamase inhibitor, alone or associated, against clinical strains of Acinetobacter baumannii Effect of combination with aminoglycosides. J. Antimicrob. Chemother. 36: 619-629. 4. Vila, J., A. Marcos, F. Marco, S. Abdalla, Y. Vergara, R. Reig, R. Gomezlus, T.J. Deanta. 1993. In vitro antimicrobial production of beta-lactamases, aminoglycoside-modifying enzymes, and chloramphenicol acetyltransferase by and susceptibility of clinical isolates of Acinetobacter baumannii. Antimicrob. Agents. Chemother. 37: 138-141. 5. Buisson, Y., G.T. Vannhieu, L. Ginot, P. Bouvet, H. Schill, L. Driot, M. Meyran, 1990. Nosocomial outbreaks due to amikacin-resistant tobramycin-sensitive Acinetobacter species Correlation with amikacin usage. J. Hosp. Infect. 15:83-93. 6. Lambert, T., G. Gerbaud, P. Bouvet, J.F. Vieu, P. Courvalin. 1990. Dissemination of amikacin resistance gene aph6 in Acinetobacter spp. Antimicrob. Agents. Chemother. 34: 1244-1248. 7. Park Y.K., S.I. Jung, K.H. Park, H.S. Cheong, K.R. Peck, J.H. Song, K.S. Ko. 2009. Independent emergence of colistin-resistant Acinetobacter spp. isolates from Korea. Diagn. Microbiol. Infect. Dis. 64: 43-51. 8. Gordon N.C., D. Wareham. 2010. Multidrug-resistant Acinetobacter baumannii: mechanisms of virulence and resistance. Intern. J. Antimicrob. Agents. 35:219-226. 9. Nemec A., L. Krizova, M. Maixnerova, T.J. van der Reijden, P. Deschaght, V. Passet, M. Vaneechoutte, S. Brisse, L. Dijkshoorn. 2011. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU). Res. Microbiol. 162: 393-404.
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10. Turton J.F., J. Shah, C. Ozongwu, R. Pike. 2010. Incidence of Acinetobacter species other than A. baumannii among clinical isolates of Acinetobacter: evidence for emerging species. J. Clin. Microbiol. 48: 1445-1449. 11. Nemec, A., T. De Baere, I. Tjernberg, M. Vaneechoutte, T.J.K. van der Reijden, L. Dijkshoorn. 2001. Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 51:1891-1899. 12. Rodriguez-Bao J., S. Mart, A. Ribera, F. Fernndez-Cuenca, L. Dijkshoorn, A. Nemec, M. Pujol, J. Vila. 2006. Nosocomial bacteremia due to an as yet unclassified Acinetobacter genomic species 17-like strain. J. Clin. Microbiol. 44: 1587-1589. 13. Nemec A., L. Dijkshoorn, I. Cleenwerck, T. De Baere, D. Janssens, T.J. Van Der Reijden, P. Jezek, M. Vaneechoutte. 2003. Acinetobacter parvus sp. nov., a small-colony-forming species isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 53: 1563-7. 14. Hung Y.T., Y.T. Lee, L.J. Huang, T.L. Chen, K.W. Yu, C.P. Fung, W.L. Cho, C.Y. Liu. 2009. Clinical characteristics of patients with Acinetobacter junii infection. J. Microbiol. Immunol. Infect. 242:47-53. 15. Nemec A., M. Muslek, O. Sedo, T. De Baere, M. Maixnerov, T.J. van der Reijden, Z. Zdrhal, M. Vaneechoutte, L. Dijkshoorn. 2010. Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae 14 sp. nov., to accommodate Acinetobacter genomic species 10 and 11, respectively. Int. J. Syst. Evol. Microbiol. 60: 896-903. 16. Fournier, P.E., D. Vallenet, V. Barbe, S. Audic, H. Ogata, L. Poirel, H. Richet, C. Robert, S. Mangenot, C. Abergel, P. Nordmann, J. Weissenbach, D. Raoult, and J.M. Claverie. 2006. Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet. 2:6272. 17. Adams, M.D., K. Goglin, N. Molyneaux, K.M. Hujer, H. Lavender, J.J. Jamison, I.J. MacDonald, K.M. Martin, T. Russo, A.A. Campagnari, A.M. Hujer, R.A. Bonomo, S.R. Gill. 2008. Comparative genome sequence analysis of multidrug-resistant Acinetobacter baumannii. J. Bacteriol. 190: 80538064. 18. Gerner-Smidt P., I. Tjernberg. 1993. Acinetobacter in Denmark: II. Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. APMIS. 101: 826-832. 19. Wisplinghoff H., T. Paulus, M. Lugenheim, D. Stefanik, P.G. Higgins, M.B. Edmond, R.P. Wenzel, H. Seifert. 2011. Nosocomial bloodstream infections due to Acinetobacter baumannii, Acinetobacter pittii and Acinetobacter nosocomialis in the United States. J Infect. [Epub ahead of print] 20. Bertini, A., L. Poirel, P.D. Mugnier, L. Villa, P. Nordmann, A. Carattoli. 2010. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54:41684177. 21. Adams, M.D., E.R. Chan, N.D. Molyneaux, R.A. Bonomo. 2010 Genomewide Analysis of Divergence of Antibiotic Resistance Determinants in Closely Related Isolates of Acinetobacter baumannii. Antimicrob. Agents. Chemother. 54: 3569-3577. 22. Smits, T.H.M., S.B. Balada, B. Witholt, J.B. van Beilen. 2002. Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria. J. Bacteriol. 184: 1733-1742. 23. Di Cello, F., M. Pepi, F. Baldi, R. Fani. 1997. Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus. Res. Microbiol. 3: 237-249. 24. Ratajczak, A., W. Geissdorfer, W. Hillen. 1998. Expression of alkane hydroxylase from Acinetobacter sp. strain ADP1 is induced by a broad range of n-alkanes and requires the transcriptional activator AlkR. J. bacteriol. 180: 5822-5827. 25. Tani, A., T. Ishige, Y. Sakai, Y., N. Kato. 2001. Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp strain M-1. J. Bacteriol. 183: 1819-1823. 26. Mengoni, A., S. Ricci, M. Brilli, F. Baldi, R. Fani. 2007. Sequencing and analysis of plasmids pAV1 and pAV2 of Acinetobacter venetianus VE-C3 involved in diesel fuel degradation. Ann. Microbiol.
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57: 521-526. 27. Throne-Holst, M., A. Wentzel, T.E. Ellingsen, H.K. Kotlar, S.B. Zotchev. 2007. Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp strain DSM 17874. Appl. Environ. Microbiol. 73: 3327-3332. 28. Diancourt L., V. Passet, A. Nemec, L. Dijkshoorn, S. Brisse. 2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One. 5: e10034. 29. Nemec, A., M. Muslek, O. edo, T. De Baere, M. Maixnerov, T.J.K. van der Reijden, Z. Zdrha, M. Vaneechoutte, L. Dijkshoorn. 2010. Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to accommodate Acinetobacter genomic species 10 and 11, respectively. Int. J. Syst. Evol. Microbiol. 60: 896-903. 30. Loubinoux, J., L. Mihaila-Amrouche, A. Le Fleche, E. Pigne, G. Huchon, P.A.D. Grimont, A. Bouvet. 2003. Bacteremia caused by Acinetobacter ursingii. J. Clin. Microbiol. 41: 1337-1338.

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